CENPA functions as a transcriptional regulator to promote hepatocellular carcinoma progression via cooperating with YY1

The centromere proteins (CENPs), a critical mitosis-related protein complexes, are involved in the kinetochore assembly and chromosome segregation. In this study, we identified that CENPA was significantly up-regulated in HCC and highly expressed CENPA correlated with poor prognosis for HCC patients. Knockdown of CENPA inhibited HCC cell proliferation and tumor growth in vitro and in vivo. Mechanistically, CENPA transcriptionally activated and cooperated with YY1 to drive the expression of cyclin D1 (CCND1) and neuropilin 2 (NRP2). Moreover, we identified that CENPA can be lactylated at lysine 124 (K124). The lactylation of CENPA at K124 promotes CENPA activation, leading to enhanced expression of its target genes. In summary, CENPA function as a transcriptional regulator to promote HCC via cooperating with YY1. Targeting the CENPA-YY1-CCND1/NRP2 axis may provide candidate therapeutic targets for HCC.


Immunohistochemistry (IHC)
Immunohistochemistry staining for tissues was performed by using the polymer HRP detection system (Zhongshan Goldenbridge Biotechnology).The tissue slices' paraffin sections underwent dewaxing, antigen retrieval with 0.01 M sodium citrate buffer (pH 6.0), incubation with 3% H2O2 at room temperature for 15 min to lessen non-specific staining, and then blocking with 5% bovine serum albumin for 60 min.Then incubated with the relevent primary antibodies overnight at 4℃ in a humidified environment, followed by a 45 min incubation with HRP conjugated secondary antibody at room temperature.The antibody binding was then detected using DAB, and the cells were counterstained with hematoxylin.For each IHC test, appropriate positive and negative controls were provided.The staining intensity and proportion of positive stained tumor cells were used to score the immunohistochemical staining.The staining intensity scoring rules were as follows: 0 points (Negative); 1 point (Light brown); 2 points (Brown); 3 points (Dark brown).The stained positive cells scoring rules were as follows: score 0 denotes less than 10%, score 1 denotes 10~25%, score 2 denotes 26~50%, score 3 denotes 51~75% and score 4 denotes more than 75% of positive stained tumor cells.
A total score of <6 and ≥6 was defined as negative and positive, respectively.

Plasmid construction, transfection, plasmid and siRNA
The full-length CDSs of CENPA and YY1 were purchased from Addgene.Wuhan Tsingke Company synthesized the K124R site mutation plasmid of CENPA.CENPA and YY1 were ligated into the expression vectors pcDNA3.1,pcDNA3.1-Flag,and pcDNA3.1-HA,respectively.For HEK293T cells, transfection with plasmids was performed using polyethyleneimine (PEI, Invitrogen, USA) at a plasmid: PEI ratio of 1: 4. For HCC cell lines, transfection with plasmids was performed using Lipofectamine 3000 (Thermo Fisher) and P3000 (Thermo Fisher) at a plasmid: Lipofectamine 3000: p3000 ratio of 1: 1.5: 2. HCC cell lines were transfected with siRNA at a final concentration of 20nM using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instruction.As a control, a negative control siRNA (NC) was used.The transfection efficiency was assessed 48 hours after transfection using a western blotting test.The sequences of siRNA are listed in Table S3.

Lentivirus-transduced stable cell lines
In order to construct gene over-expression or knockdown cells lines, we cloned the CDS region of CENPA into pLenti-CMV-Puro plasmid (Addgene #17448) or two short hairpin RNA (shRNA) against CENPA or a scramble sequence (Table S3) into the pLKO.1-TRCvector (Addgene #10879).Transient co-transfection of HEK293T cells with a lentiviral transducing vector and packaging vectors pMD2.G and psPAX2 was used to construct a recombinant lentivirus (lentivirus vector (8μg), pMD2.g (2.8μg), psPAX2 (9μg) and PEI (80μL)).Viral supernatant was then collected 48 hours after transfection.After co-cultured with lentivirus supernatant for 24 hours, HCC cells were selected and enriched by puromycin treatment at a final concentration of 10μg/ml in a culture medium.The effectiveness of CENPA overexpression or knockdown in HCC cells was assessed by western blotting assay.

EdU incorporation assay
The capacity of cells to replicate DNA was tested using a Cell-Light™ EdU Apollo567 In Vitro Imaging Kit (Ribobio, Guangzhou, China) according to the manufacturer's instructions.Briefly, the same amounts of HCC cells were seeded in 96well plate (10000 cells/well).After 12h, the media was withdrawn and 50μM EdU solutions (diluted in DMEM) were given to the cells, which were then incubated at 37°C for 2h.After removing the EdU solutions and washing 1~2 times, the cells then fixed with 4% paraformaldehyde and incubated with 0.5% TritonX-100 for 5min.The ells were then stained for 30 min with 100μl 1x Apollo solution and the nucleus with DAPI.Image was captured with EVOS FL auto imaging system (Life Technologies, USA).

Cell counting kit-8 (CCK-8) method
The viability of the cells was determined using a CCK-8 assay kit (CCK-8, Wuhan Promoter Biological CO., LTD.#P5090).For 24h, cells were grown in 96-well plates at 1000-1500 cells per well.The media was then with draw, and the cells were grown for 1 hour at 37°C with a 10% CCK-8 solution (diluted in serum-free DMEM).The optical density (OD) value of the cell was determined, and the cell proliferation curve was constructed by measuring the 450 nm absorbance at each indicated time point.

Colony formation assays
For the colony formation assay, HCC cells were seeded in 6-well plates at a density of 1000-2000 cells per well.The medium was refreshed every 3 days.After 2 weeks, the colonies were washed in phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and imaged using an optical microscope.

Cell cycle assay
HCC cells were seeded in 6-well plates, after reaching the logarithmic stage of growth.HCC cells were digested and washed.The cells were resuspended in 75% ethanol for 24 h at 4°C, then the DNA was stained with propidium iodide (PI) at room temperature for 30 min in the dark.The indicated cell cycle phase of HCC cells was analyzed by flow cytometry (BD FACS Calibur, BD Biosciences, San Diego, CA, USA).

Animal experiments
The entire protocol was carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 86-23, revised 1985) and was authorized by Tongji Hospital's Committee on the Ethics of Animal Experiments (TJH-20210312).
Male BALB/C nude mice (4 weeks old) were purchased from Beijing HFK Bioscience Co. Ltd. (China) and were kept in a specific-pathogen-free (SPF) environment.In the subcutaneous tumor formation assay, we injected 1×10 6 HCC cells in 100μl of serumfree DMED subcutaneously into the flanks of nude mice (5 mice per group).The tumor volumes were assessed every 3 days, and tumor weight was recorded after 4-8 weeks of sacrifice.The volume was calculated according to below: (volume, mm 3 ) = 0.5 × L (length, mm) ×W 2 (width, mm 2 ).To evaluate the tumor growth in vivo, we injected 1×10 6 HCC cells diluted 30μl serum-free DMEM into the nude mice livers (5 mice per group).4 weeks later after transplantation, harvested all mice tumor.Tumor volumes and tumor weights were measured as indicated above.In the tumor formation assay, we injected 1×10 6 HCC cells in 30μl of serum-free DMED subcutaneously into the liver of mice (5 mice per group), and tumor weight was recorded after 4 weeks of sacrifice.The 2-DG group, mice were received intraperitoneal injection of 400mg/kg every 3 days.

Co-immunoprecipitation (Co-IP), Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis, silver staining
Harvested HCC cells were suspended in 1 ml IP-lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, and protease inhibitor cocktail, pH 7.4).The supernatant was subjected to ultrasonication, and centrifugation was performed at 12000g for 15 min at 4°C.After that, Protein G agarose beads were used to preabsorbed the lysates of cells without or with stable transfection of tagged constructs for 2h at 4°C.Then, incubated the supernatant with the target antibody or IgG overnight in a 4°C shaker.The second day, Protein G agarose beads were added to the antibody/supernatant complex for 2 hours at 4°C.After extensive washing 5-6 times with IP-wash buffer (50 mM Tris-Cl, 300 mM NaCl, 1% Triton X-100, 1 mM EDTA, pH7.4), the samples were analyzed using western blotting to identify the potential interacting proteins.The products of co-IP assays were sent to NOVOGENE Company Limited (Beijing, China) for protein peptide detection.A Silver Stain Kit (Beyotime, Shanghai) was used to detect the expression of CENPA and its interacting proteins.

Immunofluorescence (IF)
Wild-type HCC cell lines were seeded in 24-well plate containing glass slides for 12 h.The cells were then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% TritonX-100 for 5 minutes at room temperature, and sealed in PBS containing 5% BSA.The primary antibodies were added overnight at 4°C.Cells were incubated with secondary antibodies from different species for 1 h at room temperature.
Then use DAPI to counterstained nuclei for 5 min at room temperature.A Zeiss LSM scanning microscopy (Carl Zeiss, Germany).

Cellular Fractionation
HCC cells were washed with PBS and scraped in 1 ml ice-cold PBS.Collect the cells and pellet cells at 130 x g in a refrigerated centrifuge at 4 °C for 3 min.Pellet cells were suspended in 5 volumes of ice-cold E1 buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH8.0, 10% glycerol, 0.5% NP-40, 0.25% triton X-100, 1 mM DTT, 1x protease inhibitor cocktail).Then centrifuge and collect the supernatant as cytoplasm fraction, and the pellet was washed by E1 buffer for 2 times.After centrifugation at 1100 x g at 4 °C for 3 min, discarded the supernatant and resuspended the pellet by 2 volumes of ice-cold E2 buffer (10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA pH8.0, 0.5mM EGTA pH8.0, 1x protease inhibitor cocktail).Then centrifuge and collect the supernatant as nucleus fraction, and the pellet was washed by E2 buffer for 2 times.After centrifugation at 1100 x g at 4 °C for 3 min, discarded the supernatant and resuspended the pellet by E3 buffer (500mM Tris-HCl, 500mM NaCl, 1x protease inhibitor cocktail).The final solution (chromatin fraction) was sonicated for 5 min, and all fraction were centrifuged at 1600 x g at 4 ℃ for 10min and for western blot analysis.

Dual-luciferase reporter gene assay
The genomic promoter sequence, which was cloned into the pGL4.17[luc/Neo] plasmid (Promega, USA) between -2000 and +100 base pairs (bp) from the cloned the transcription start site (TSS) were synthesized by Wuhan Tsingke Company.The sitedirected mutagenesis promoter sequence was also synthesized by Wuhan Tsingke Company.HCC cell lines were seeded into 24-well plates, then 480ng of pGL4.17 plasmid and 2ng of pRL-Renilla-luciferase plasmid were cotransfected into each well.
After 48 h, cells were lysed in passive lysis buffer (Promega, USA) for 30 min and collected.The Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was used in accordance with the manufacturer's instructions to detect luciferase activity.
Renilla activity was used to standardize the relative luciferase activity.

Chromatin immunoprecipitation (ChIP)
The SimpleChIP® Plus Sonication Chromatin IP Kit (Cell Signaling Technology #56383) was used for the chromatin immunoprecipitation (ChIP) assays.Briefly, HEK293T cells or HCC cell line were seeded in 15 cm dishes.The cells were then fixed, and the cell lysis was collected.The sonicator microprobe was used with the cell lysates for 4 rounds of 10 min, 1s pluses at output level 6.The shared chromatin was first incubated either with antibodies against CENPA or YY1 or with normal rabbit IgG overnight at 4°C, and followed by a 2h incubation with beads at the same temperature.
The beads were washed with wash buffer four times, and then DNA was isolated for sequencing service or quantitative PCR.Sequencing service was provided by Bioyi Biotechnology Co., Ltd.Wuhan, China.The ChIP primers of CENPA and YY1 target genes are listed in Table S3.

Figure S4 .
Figure S4.YY1 promotes HCC growth, proliferation, and cell cycle progression in

Figure S5 .
Figure S5.YY1 may serve as the target genes regulated by CENPA.